carbohydrate synthesis part 2: solution and solid phase chemical synthesis
DESCRIPTION
Carbohydrate Synthesis Part 2: Solution and solid phase chemical synthesis. Chemoenzymatic synthesis. “Essentials of Glycobiology” 3 June 2004 Michael VanNieuwenhze/Nathaniel Finney Dept. of Chemistry and Biochemistry UCSD [email protected]. Lecture Outline - PowerPoint PPT PresentationTRANSCRIPT
Carbohydrate SynthesisPart 2: Solution and solid phase chemical synthesis.
Chemoenzymatic synthesis.
“Essentials of Glycobiology”3 June 2004
Michael VanNieuwenhze/Nathaniel FinneyDept. of Chemistry and Biochemistry
Lecture Outline
1. Iterative solution phase synthesis by Danishefsky’s glycal method.
2. Identity of glycosyl substituents alters the reactivity of glycosyl donors: Exploitation in Wong’s solution phase Optimer methodology.
3. Solid phase carbohydrate synthesis possesses many of the same advantages of solid phase peptide and oligonucleotide synthesis: Automated oligosaccharide synthesis.
4. Chemoenzymatic synthesis of oligosaccharides and glycoconjugates: Complementary to chemical methods; narrower is scope but more elegant and efficient in execution.
Iterative Solution Phase Synthesis with Glycals
Now that we’ve discussed the basics of chemical glycosylation, let’s look at more complex synthetic challenges. The Lex-Ley nonasaccharide is not readily available from natural sources, but could be valuable for, e.g., developing anticancer vaccines.
Remarkably, this oligosaccharide can ultimately be prepared from just 3 glycal precursors.
O
HO OH
OH
O
OHO
HO
OHO
NHAc
OH
OO
OH
HO
O
OHO
NHAc
OH
OO
OOH
HO
O
OH
O
OHHO
HO
OO
HO OH
OH
O
O
HO
OH
OHOH
Glycals for the Solution Phase Synthesis of Lex-Ley
The 3 glycals (in general form):
Glycal method - quick reminder:
OOOPRO
ROHOR*O
OPOHO
PO
OP
A B C
P = protecting groupR = P or HR' = nitrogen protecting groupR* = unique hydroxy protecting group and fucosylation site
O
OBnBnO
OBnO
OBnBnO
OBnO
OBnBnO OH
OBnOR
OROH"DMDO"
ZnCl2
First Stage Glycal Couplings
O OOPO
OP
OH
PO
PO
OPO OO
R*O
OP
OH
PO
PO
OP
A + CA + B
O OO
R*O
OP
OP
PO
PO
OPO OO
PO
OP
OR
RO
HO
OP
O
OP
RO
PO
OPO
NHR'R*O
OP
OO OO
PO
OP
OP
RO
O
OP
+
2 steps1 step
2 steps
O OOR*O
OPPO
PO
OP
R*O
1 step
Second Stage Glycal Couplings
O OOR*O
OPPO
PO
OPO
OP
RO
HO
OPO
NHR'R*O
OP
OO OO
PO
OP
OR
RO
O
OP
+
steps
O
OR*
RO
RO
OPO
NHR'R*O
OP
OO
OP
PO
O
OPO
NHR'R*O
OP
OO OO
PO
OP
OP
PO
O
OP
R*O
O
PO OP
OP
O
ORO
RO
OPO
NHR'
OP
OO
OP
PO
O
OPO
NHR'
OP
OO OO
PO
OP
OP
PO
O
OP
steps
O
OPPO
PO
OO
PO OP
OP
O
Glycal Couplings - Summary
Pro: Synthesis of important and previously inaccessible oligosaccharide achieved.
Reaction conditions are mild and general.
Better than alternatives available at the time.
Con: Very labor intensive (~1.5 year to develop, 6 months to repeat).
Isolation, purification of intermediates difficult and time consuming.
Overall yield < 1%.
Protecting Groups Alter Glycosyl Donor Reactivity
For glycosylation with glycosyl cations, would predict that two of the things influencing the rate of glycosylation would be:
1. The rate at which the glycosyl cation is generated.
2. The reactivity of the glycosyl cation itself.
Specifically:
O
ORRO OR
ORX O
ORRO OR
OR
activation
Protecting groups(R) may influence
reactivity of X.
Variations in structure of Xmay alter rate of activation.
Protecting groups(R) will influence
reactivity of cation.
More Specific Predictions of Reactivity
Focusing on the cation itself, would predict that electron donating protecting groups (Bn, PMB, TBS, e.g.) should stabilize a glycosyl cation, while electron withdrawing protecting groups (Ac, Bz, Piv - remember the carbonyl dipole, C=O +C–O–) should destabilize a glycosyl cation.
OO
OO O
H3C
H3C
CH3
CH3OO
O
O
A glycosyl cation withelectron withdrawingacetate protecting groups...
OO
OO O
...should be less stable/more reactive than the
benzyl protected analog.
Wong’s Optimer Method - Retrosynthesis
The Wong Group (Scripps Research Institute) has prepared hundreds of mono and disaccharides with different protecting group patterns to control glycosylation rate. This means that multiple components can be combined in the same reaction: the fastest component reacts first, then the next fastest, etc.
A computer program (Optimer) has been written that knows the relative reactivities of all of these potential reactants. It can carry out retrosynthetic analyses of complex carbohydrates and suggest a set of reactants that would allow synthesis of the oligosaccharide in one single reaction.
In addition to condensing multiple reactions into one, this dramatically simplifies the laborious and inefficient processes of isolation and purification.
Wong’s Optimer Method - Illustration
OOH OH
HOO
OO
OH OH
OO
OHOH
NHAc HO OO
OH OHOH O OHO
OH
OH
OHO
OH
OHORGlobo H
Optimer says that...
...can be prepared from these three components.
OOBn
OBn
BnOOH
OO
OBzOBz
OO
ONBz
TrocHN OClBn
NBzO OHO
OBnO
OBn
OBnO
BnO
OBn
OBnOR
O
OBnOBnOBn
STol
STolA
B C
(STol ~ SPh; NBz, Troc = similar to Ac; ClBn similar to Bn.)
Wong’s Optimer Method - Illustration
OOBn
OBn
BnOO
OO
OBzOBz
OO
ONBz
TrocHN ClBnO OO
OBnOBnOBn O
OBnO
OBn
OBnO
BnO
OBn
OBnOR
NBzO
OOBn
OBn
BnOOH
OO
OBzOBz
OO
ONBz
TrocHN OClBn
NBzO OHO
OBnO
OBn
OBnO
BnO
OBn
OBnOR
O
OBnOBnOBn
STol
STolA
B C
DMTSTCH2Cl2–78 °C to RT
one pot62 % yield (!)
Wong’s Optimer Method - Summary
Pro: Remarkably efficient and predictable assembly of complex carbohydrates in a single reaction. (Compare Globo H synthesis to that of Lex-Ley shown previously.)
Minimizes synthetic effort.
Dramatically reduces labor associated wiith isolation/purification.
Con: Requires access to a very large number of complex precursors.
Synthetic method still not accessible to non-chemists.
Solid Phase Synthesis of Oligomeric Biomolecules
Everyone probably knows solid phase deoxyoligo synthesis:
ODMTOB1
O
O
solid support
H+
DMT
OHOB1
O
O
5'
3'
ODMTOB2
OP N
R
RONC
OOB1
O
O
ODMTOB2
OP
ONC
OOB1
O
O
ODMTOB2
OP
ONC
Oxidation
OH+
Deprotection
Coupling
I2, water
activator+
Advantages of Solid Phase Synthesis
Here are the most relevant advantages of solid phase peptide and oligonucleotide synthesis relative to solution-phase:
1. Improved yields and purities. Use of large excess of solution phase reagents can drive reactions to completion, as can repetition of reaction cycle.
2. Simplified isolation and purification. Multiple reactions require only a single isolation and purification, at the very end. (Of course, this means the chemistry has to be very efficient….)
3. No problems with solubility/precipitation. Material is site isolated (no chance for aggregation) on an insoluble solid support.
4. Automation!
Solid Phase Oligosaccharide Synthesis
Synthetic chemistry has finally advanced to the point that solid phase oligosaccharide synthesis is also feasible, and can now be automated.
HOO
2) H2NNH2
TMSOTf
2) NaOMe/MeOH
O
BnO
OBnO PivO
OBn
PO
OBuOBu
TMSOTf
1) 1)
LevO OBnO
BnO OAc
O
NH
CCl3
BnO OBnO
BnO OAc
O
NH
CCl3
TMSOTf
1)
BnO OBnO
BnO OAc
O
NH
CCl3
TMSOTf
1)
2) Cleavage from resin.2) NaOMe/MeOHO
BnO
BnO OO
BnO
OBnO PivO
OBn
BnO OBnO
BnO O
BnO OBnO
BnO OAc
O
Protected Leishmaniaantigen. Automatedsynthesis provides
42% in 9 hours!
Solid Phase Oligosaccharide Synthesis - Summary
Pro: Remarkably efficient synthesis of complex oligosaccharides.
Automated solid phase synthesis faster (often by > 10x) and higher yielding than corresponding solution phase syntheses.
Automation makes synthetic methodology accessible to non-chemists.
Con: Many glycosidic linkages of interest still inaccessible. (This is true of all chemical glycosylation approaches.)
Even with automation, a bewildering number of specialized reactants are still required. Not clear how this will be solved, although it must be if synthesizers are to become commercially available/viable.
Chemoenzymatic Synthesis of Oligosaccharides
As an alternative to chemical synthesis, many biochemists and bioorganic chemists have explored the use of glycosidases and glycosyltransferases in the synthesis of oligosaccharides and glycoconjugates.
The appeal of this approach is obvious: Nature has already figured out how to make all of the naturally occurring oligosaccharides, and if we could borrow from Her toolbox we’d save a lot of time and effort.
The use of glycosyltransferases and glycosidases have strengths and weaknesses that are in many ways complementary to those of chemical synthesis. Let’s look at a few examples before we discuss the Pros and Cons of the approach.
Synthetic Application of Glycosyltransferases
A comparison of the enzymatic and chemical synthesis of tetra- and pentasaccharide cell-surface epitopes from Neisseria meningitidis (a causative agent of meningitis) is instructive.
O
HO
OHOH
HO OSPh
HO
OHO
HO
O
HO
OHOH
OSPh
HO
OHO
HO
OO
AcHN
OHHO
HO
GlcNAc transferaseUDP-GlcNAc
96% (28 mg)
O
HO
OHOH
HO OSPh
HO
OHO
HO
O
BzO
OBzOH
OSPh
BzO
OBzO
BzOHO
3 steps, 55%
Chemoenzymatic synthesisof meningitis epitope:
Chemical synthesisof meningitis epitope:
A B
Application of Glycosyltransferases
O
HO
OHOH
OSPh
HO
OHO
HO
OO
AcHN
OH
HO
O
HO
OHOH
OHO
O
HO
OHOH
OSPh
HO
OHO
HO
OO
AcHN
OH
HO
O
HO
OHOH
OO
CO2H
AcHNHO
HOHO
OH
O
UDP-Glc epimerase-Gal transferase fusion
UDP-Glc
CMP-Neu5Ac synthase-transferase fusion
CTP, Neu5Ac
96% (24 mg)
97% (30 mg)
O
BzO
OBzOH
OSPh
BzO
OBzO
BzO
OO
PhthalN
OAc
AcO
O
AcO
OAcOAc
OAcO
O
HO
OHOH
OSPh
HO
OHO
HO
OO
AcHN
OH
HO
O
HO
OHOH
OHO
2 steps, 66% (165 mg)
O
PhthalN
OAc
AcO
O
AcO
OAcOAc
OAcO
O CCl3
NH
1 step, 40% (260 mg)
9 steps fromlactoseA B
Chemoenzymatic synthesisof meningitis epitope (cont.):
Chemical synthesisof meningitis epitope (cont.):
Application of Glycosyltransferases - Summary
Pros:
Excellent yield with complete regio- and stereoselectivity.No protecting groups needed.Sialyltransferases allow facile sialylation. (This is the hardest glycosylation to carry out by chemical methods.)
Reactions can be carried out in water.
Cons:
Most requisite enzymes are not readily available, and those that are available are expensive.
Regio/stereoselectivity means that substrate scope is limited and a unique enzyme is needed for almost every reaction.
Nucleotide sugar donors are very expensive and/or unstable.Scale is often limited by enzyme avilability/volumetric productivity. (A large amount of enzyme produces only a small amount of sugar by weight.)
Application of Glycosyltransferases - Final Note
Although issues of enzyme cost/availability/substrate scope are likely to remain unsolved for some time, the issues of scale and nucleotide donor cost/availability can sometimes be overcome:
OOH
AcHN
OHOH
OO
HO
OH
HO
OH
OOH
AcHN
OHOH
OO
HO
OHOH
O
CO2H
AcHNHO
HOHO
OH
O
CMP-Neu5Ac
CMP sialic acid + phosphoenolpyruvate
pyruvate
fusionprotein
59 g (as monohydrate)
68 g, 68 % yield
fusionprotein
Synthetic Application of Glycosidases
Another chemoenzymatic approach is to employ glycosidase. While these enzymes normally cleave glycosidic linkages, they can be coerced to “run backwards” (to some extent) in the presence of an appropriate glycosyl donor.
OO
HO
OH
HO
OH
NO2
Gal-β-PNP
OOR
HO
OH
HO
OH β-galactosidase
HO NO2
PNP
β-galactosidase
OOH
HO
OH
HO
OH
+ ROH
+ ROH OOR
HO
OH
HO
OH
Application of Glycosidases
The synthesis of 2 Tn-antigen epitopes is illustrative:
O
OAcHN
OHOH
AcHN
Me
O
OMe
HO
O
OAcHN
OHOH
AcHN
Me
O
OMe
OO
HO
OHOH
O
CO2H
AcHNHO
HOHO
OH
O
β-galactosidase O
OAcHN
OHOH
AcHN
Me
O
OMe
OO
HO
OH
HO
OHOO
HO
OH
HO
OH
NO2
HO NO2
Gal-β-PNP
PNP
+
O
OAcHN
OHOH
AcHN
Me
O
OMe
OO
HO
OH
HO
Neu5Ac-a-PNP
PNP
PNP
Neu5Ac-a-PNP
sialidasesialidase
OCO2H
AcHN
HO
HO OH
OH
O
12% (5 m g) 15% (5 m g)
22% (13 m g)
Application of Glycosidases - Summary
Pros:
Many glycosidases available (esp. in comparison to glycosyltransferases).
Glycosidases are less expensive and more stable than transferases.
Specificity is generally relaxed rel. to transferases, allowing broader substrate scope.
Generally good regio- and stereoselectivity.No protecting groups.
Cons:
Enzymatic glycosylations still less scalable that chemical reactions.
Volumetric productivity still low.Yields much lower than with transferases - glycosidase activity (which degrades the product) competes with glycosylation.
Chemical v. Chemoenzymatic Synthesis - Summary
The use of glycosyltransferases and glycosidases have strengths and weaknesses that are in many ways complementary to those of chemical synthesis. Chemical synthesis provides flexibility that allows the preparation of diverse natural and unnatural structures, but requires the extensive use of protecting groups and preparation of specialized precursor compounds. In contrast, enzymes are typically much less flexible and/or available, but do not require the use of protecting groups or the preparation of elaborate precursors. In the near term it appears likely that both approaches will remain in use: indeed, a great many of the most successful applications of enzymes in oligosaccharide synthesis have been in “chemoenzymatic” syntheses, relying on a hybrid of chemical and enzymatic methods that typically begins with chemical synthesis and ends with enzymatic elaboration.